The highly specific occurrence of selenocysteine in several selenium-dependent enzymes is now well established and in at least two cases its cotranslational insertion is directed by the opal stop codon, UGA. For this process serine is esterified to a special tRNA having an anticodon complimentary to UGA and then the seryl-tRNA is converted to selenocysteinyl-tRNA which is used for protein synthesis. Selenomethionine, in contrast, is inserted ubiquitously into many proteins in animals, plants, and bacteria as a non-specific substitute for methionine; its specific occurrence has not been documented. Thus, the reported presence of selenocysteine in a Salmonella typhimurium mutant defective in synthesis of specific selenoenzymes (and also seleno-tRNAs) suggested this might be a case of random replacement of cysteine in proteins with selenocysteine. Analysis of this mutant and also a pleotrophically similar Escherichia coli mutant revealed that both organisms retain the ability to synthesize selenocysteine and selenomethionine and that these selenoamino acids are associated with many proteins of the cells in a form that requires strong acid hydrolysis or proteolytic digestion for liberation. The nature of the mutation that prevents normal highly specific incorporation of selenium into formate dehydrogenases and into tRNAs and instead appears to result in selenocysteine inclusion in many proteins has special significance regarding selenium toxicity and will be investigated in greater detail. Replacement of the essential selenocysteine residue in a formate dehydrogenase of E. coli with a cysteine residue by site-directed mutagenesis resulted in an enzyme with markedly lower catalytic activity. Procedures have been developed for purification of both forms of the enzyme in order to compare accurately the relative effectiveness of a selenol versus a thiol as a redox center in an enzyme. Both enzymes are extremely oxygen labile and difficult to isolate. The sulfur enzyme is estimated to be 10 or at most 20% as active as the selenium form.